1. Field of the Invention
This invention relates generally to compositions and methods of amplifying and cloning nucleic acid sequences. Specifically, the invention is directed to compositions and methods using reverse transcriptase, polymerase chain reaction, and cloning vectors for the production of full length nucleic acid sequences from viral genomes.
2. Description of the Related Art
Long RT-PCR (LRP) amplification of RNA templates is sometimes difficult compared to long PCR of DNA templates. There exists a long felt need for a reliable method of replicating and amplifying sequences from long RNA templates. Polymerase chain reaction (PCR) is an indispensable technique in biomedical research. With known primer sequences, it can easily amplify a DNA target less than 3 kb but it has diminished power when the target is larger than 3 kb. In 1994, Barnes et al first hypothesized that the inability to amplify large DNA fragments was due to the misincorporation of nucleotides by most thermostable DNA polymerases, which resulted in premature termination of PCR [1]. Based on this hypothesis, mixed polymerases, one of which has 3′ to 5′ exonuclease “proofreading” activity to correct the misincorporation, have successfully amplified DNA targets up to 42 kb [2]. However, there has been limited success in applying this concept to the amplification of large RNA genomes that require the reverse transcription (RT) step prior to PCR amplification. Compared to the amplification of DNA targets, it is reasonable to hypothesize that the RT step is of crucial importance during long RT-PCR (LRP) performance when taking into account the following characteristics. First, in most situations, the solution buffers are not compatible between RT and PCR. Only part of the RT reaction can be used for subsequent PCR and thus reduces the sensitivity dramatically. Second, most RT enzymes have an inhibitory role for thermostable DNA polymerases [3]. Third, RT is conducted at temperatures ranging from 37° C. to 50° C. at which the RNA template may retain its secondary structure that makes RT stop prematurely. Such situations are even more challenging when trying to amplify full-length hepatitis C virus (HCV) genome, a positive sense single-strand RNA virus in the family of flavirividae. There is extensive secondary structure along the entire HCV genome [4-6]. Furthermore, HCV cannot be cultured in vitro. The only source of RNA template for LRP is clinical samples in which HCV has a low titer.
The inventors have sought to investigate each step of the LRP procedure and developed a robust protocol for the efficient amplification and cloning of near full-length HCV genome from clinical samples, and in addition estimate the sensitivity and potential PCR-mediated recombination related to this protocol.